Candidatus Phytoplasma Trifolii Research Articles

First report of a ‘Candidatus Phytoplasma trifolii’-related strain (16SrVI-A) associated with watermelon in Türkiye

First report of a ‘Candidatus Phytoplasma trifolii’-related strain (16SrVI-A) associated with watermelon in Türkiye

First report of Candidatus Phytoplasma trifolii (16SrVI-D) associated with little leaf disease of Nyctanthes arbor-tristis in the world

First report of Candidatus Phytoplasma trifolii (16SrVI-D) associated with little leaf disease of Nyctanthes arbor-tristis in the world

Seasonal and lifecycle changes in behavior affect the trapping efficiency of an insect vector, Circulifer tenellus (Hemiptera: Cicadellidae)

Abstract Accurate sampling of pests is the foundation of pest management. Choosing the best trap for pest monitoring can be complex, however, because trap performance is affected by pest preferences and behaviors. Moreover, preservation of DNA in traps is a consideration when insect specimens are used in molecular assays, such as the detection of insect-borne pathogens. We assessed the efficiency of 2 trap designs and 2 trap placements on the capture of beet leafhopper, Circulifer tenellus (Baker) (Hemiptera: Cicadellidae), vector of “Candidatus Phytoplasma trifolii” and Beet curly top virus. Trap designs included standard yellow sticky cards and 3D-printed traps that capture insects directly in a DNA preservative. We found that yellow sticky cards captured more adults than did 3D-printed traps during summer months but captured fewer adults during autumn when leafhoppers move to overwintering sites. 3D-printed traps captured more nymphs than sticky cards, regardless of season, and traps hung at ground level captured more nymphs and adults compared to traps at 1-m height. Contrary to predictions, we did not find differences between trap types in the molecular detection of Ca. P. trifolii or Beet curly top virus, perhaps because 3D-printed traps captured few leafhoppers during summer when the pathogen rates were highest. Our results suggest behavioral differences in C. tenellus trap preference based on seasonality and life stage and underscore the importance of understanding insect behaviors when choosing trap designs for pest monitoring as well as for properly interpreting trap capture data.

First Report of Lingonberry Stunted Yellows Disease of Vaccinium vitis-idaea L. associated with 'Candidatus Phytoplasma trifolii'-Related Phytoplasma Strain in Lithuania.

Lingonberries (Vaccinium vitis-idaea L.) are low-growing, evergreen shrubs of cooler, northern regions of North America and Europe. These plants produce berries that are unique in flavor, bear high economic significance, and play a vital role in maintaining the diversity of the northern ecosystems (Kowalska, 2021). In October 2023 diseased plants of lingonberry were discovered in Labanoras Forest (55°14'N 25°42'E) (Lithuania). The plants expressed symptoms of stunting, yellowing, little leaf, shortened internodes, and stem distortions. Samples (leaves) were collected and tested from ten asymptomatic and ten symptomatic lingonberry plants. Total genomic DNAs of all samples were extracted by a CTAB protocol. Extracted DNAs were used as a template in direct and nested PCRs using the universal primer pairs P1/P7 and R16F2n/R2, respectively, to amplify phytoplasma 16S rRNA gene 1.2 kb fragments (Lee et al. 1998). The primer pairs SecAFor1/SecARev3 and SecAFor2/SecARev3 were used in direct and semi-nested PCRs, respectively, to amplify phytoplasma secA genes 0.5 kb fragment (Dickinson and Hodgetts, 2013). PCR amplicons of the 16S rRNA and secA genes specific for the phytoplasmas were only obtained from all sampled symptomatic plants. Three R16F2n/R2 and three SecAFor2/SecARev3 amplicons were cloned and submitted for Sanger sequencing (Nature Research Centre, Vilnius, Lithuania by 3500 Genetic Analyser). The three 16S rDNAs as well as the three secA gene fragments were identical. The BLAST analysis (NCBI) of the obtained sequences showed a similarity percentage, ranging from 99.75% to 100% (1247-1250 bp from 1250 bp) for 16SrRNA, and 98.13% to 99,15% (473-478 bp from 482 bp) for secA amplicons, with numerous strains of 'Candidatus (Ca.) Phytoplasma (P.) trifolii' (first hit MT674293 and KR906724, respectively). Additionally, 16S rDNA sequences by using iPhyClassifier were used to create virtual RFLP pattern (Zhao et al. 2009). The generated pattern was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group VI, subgroup A. The phytoplasma strain detected in lingonberries was designated as lingonberry stunted yellows, LingbSY. Furthermore, the enzymatic RFLP analysis was performed with the 14 restriction enzymes (Lee et al., 1998), and obtained profiles were compared with virtually generated using iPhyClassifier. This yielded the same classification of detected phytoplasma to the 16SrVI-A phytoplasma subgroup. The phylogenetic analysis of both marker gene sequences revealed the same LingbSY phytoplasma classification. Selected sequences were deposited in GenBank (NCBI) with Accession No: PP237769 (16S rRNA gene) and No: PP238489 (secA gene). Phytoplasmas of 16SrI phytoplasma group were identified in lingonberries in Canada (Brochu et al. 2022). Strains of 16SrVI phytoplasma group were reported in Vaccinium myrtillus in Austria (Fernandez et al. 2007). This is the first report of 'Ca. P. trifolii' strain belonging to 16SrVI-A phytoplasma subgroup infecting lingonberry worldwide. Also, this is the first report of 16SrVI phytoplasma group in Lithuania. The presence of this phytoplasma poses a threat to the natural ecosystem and could eventually spread into agricultural settings in our country. Therefore, it's crucial to conduct surveillance for insect vectors, and assess effective control methods. Without proactive action, long term sustainability of lingonberries and their ecosystems may be jeopardized.

Hemp Abnormal Growth Is Attributed to Mono-, Di-, or Tri-Infections of Spiroplasma citri, ‘Candidatus Phytoplasma trifolii’, and Beet Curly Top Virus

As hemp ( Cannabis sativa) emerges as a commercial crop in Nevada, mild to severe abnormal growth has been observed from many plants in commercial fields throughout the growing season. Affected plants exhibited a group of symptoms typically including stunting, leaf yellowing, excessive apical branching, clusters of witches’ broom, leaf rolling upwards, and leaf mottling and mosaic. These symptoms mostly showed up together in a plant or a crop and are defined here as abnormal growth syndrome (AGS). Between 2017 and 2019, the Nevada State Plant Pathology and Molecular Diagnostic Lab received 67 symptomatic hemp samples from Nevada for diagnosis, and ‘ Candidatus Phytoplasma trifolii’ was detected in 14 samples (21%). To investigate additional biotic agents associated with AGS, PCR products generated by primers P1/P7 were cloned into a pGEM-T vector and sequenced, and Spiroplasma citri DNA was found in two samples (3%). All 67 DNA samples were further tested for beet curly top virus (BCTV), and 57 samples (85%) were found to be infected by BCTV. Twelve samples (18%) were coinfected by both ‘ Ca. Phytoplasma trifolii’ and BCTV, and two (3%) were coinfected by S. citri, ‘ Ca. Phytoplasma trifolii’, and BCTV. The findings suggest that BCTV is the most prevalent pathogen causing the hemp abnormal growth in Nevada, but ‘ Ca. Phytoplasma trifolii’ and S. citri may also contribute to the severity and complexity of symptoms. Thus, hemp abnormal growth can be attributed to single, dual, or triple infections of these three leafhopper-vectored mollicutes and virus. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Status of virus and phytoplasma diseases in carrot growing areas of Ankara and Konya provinces, Turkey

In this study, infections that may emerge from viruses and phytoplasmas were investigated in carrot cultivation fields in the Ankara and Konya provinces, which are located in the Central Anatolia Region of Turkey and have the largest agricultural areas of the country. A total of 272 symptomatic and asymptomatic carrot plants, together with their root and leaf parts, were collected from 61 fields by performing periodic field studies during the plant vegetation periods between 2018–2019. Samples were tested against carrot virus Y (CarVY), carrot red leaf virus (CtRLV), carrot red leaf virus-associated RNA (CtRLVaRNA), carrot mottle virus (CMoV), carrot torradovirus 1 (CaTV), carrot yellow leaf virus (CYLV), carrot thin leaf virus (CTLV) and cucumber mosaic virus (CMV) agents by Reverse Transcriptase-PCR (RT-PCR). To determine phytoplasmas, samples were tested using universal primers by nested PCR. Molecular tests conducted to investigate the aforementioned viruses produced negative results, but phytoplasma infections were widely found. 16SrVI-A “Candidatus Phytoplasma trifolii” and 16SrI-B “Aster yellows” subgroups were detected by in vitro/in silico RFLP and sequencing analyses. Disease incidence caused by both phytoplasma groups was 39.34%. This study determined for the first time that 16SrVI and 16SrI groups cause severe infections in the carrot fields of Turkey.

‘Candidatus Phytoplasma trifolii’ associated with faba bean phyllody in Jordan

‘Candidatus Phytoplasma trifolii’ associated with faba bean phyllody in Jordan

USED NASTED-PCR DETECTION OF PHYTOPLASMA CAUSING BIG BUD DISEASE ON TOMATO IN IRAQ

Symptoms similar to those produced by the phytoplasma disease "Big bud" were observed on tomatoes (Solanum lycopersicum L.) grown in the field in Mosul city, Iraq. Diseased plants were characterized by twisting, corrugated, yellowing or reddening of leaves. The sepals of the flowers acquired hypertrophied form, were fused together and created a bell-shaped sterile bud (phyllody) of green or anthocyanin color. The stems of the plants were lignified, and phloem necrosis was observed on the stem. In mid-September 2020, samples of 30 diseased and 2 healthy (control) tomato plants were collected from the fields. Phytoplasmas were detected by PCR and Nested-PCR in 10 diseased samples, use universal and specific primers. The results of a phylogenetic tree consisting of 27 genetic sequences of the 16 S ribosomal RNA gene of Candidatus Phytoplasma trifolii, locally isolated from Iraq/Mosul, showed a sequence identity with a high percentage of genetic similarity of 99% with Candidatus Phytoplasma trifolii 16Sr member VI-A. Clover proliferation group (16Sr VI), with various isolates from around the world.

Cas12a-Based Diagnostics for Potato Purple Top Disease Complex Associated with Infection by ‘Candidatus Phytoplasma trifolii’-Related Strains

'Candidatus Phytoplasma trifolii' is a cell wall-less phytopathogenic bacterium that infects many agriculturally important plant species such as alfalfa, clover, eggplant, pepper, potato, and tomato. The phytoplasma is responsible for repeated outbreaks of potato purple top (PPT) and potato witches' broom (PWB) that occurred along the Pacific Coast of the United States since 2002, inflicting significant economic losses. To effectively manage these phytoplasmal diseases, it is important to develop diagnostic tools for specific, sensitive, and rapid detection of the pathogens. Here we report the development of a DNA endonuclease targeted CRISPR trans reporter (DETECTR) assay that couples isothermal amplification and Cas12a transcleavage of fluorescent oligonucleotide reporter for highly sensitive and specific detection of 'Candidatus Phytoplasma trifolii'-related strains responsible for PPT and PWB. The DETECTR assay was capable of specifically detecting the 16S-23S ribosomal DNA intergenic transcribed spacer sequences from PPT- and PWB-diseased samples at the attomolar sensitivity level. Furthermore, the DETECTR strategy allows flexibility to capture assay outputs with fluorescent microplate readers or lateral flow assays for potentially high-throughput and/or field-deployable disease diagnostics.

First report of ‘Candidatus Phytoplasma trifolii’ associated with little leaf disease of Capsicum chinense from the northeast of India

King chilli or bhut jolokia (Capsicum chinense, family Solanaceae), is a variety of capsicum indigenous to northeast India. It is considered one of the hottest chillies in the world and is one of the most valuable spice crops in India, being grown for culinary and medicinal purposes. In April 2022, phytoplasma-associated symptoms such as shortening of leaves, petioles and internodes, and stunting of the whole plant were observed on C. chinense plants in Jorhat, a district in Assam, India (Figure 1). To investigate the possibility of a phytoplasma being associated with the symptoms, total DNA was isolated from leaf samples collected from six diseased and six asymptomatic plants using a CTAB protocol (Kollar et al., 1990). The DNA was analysed by direct and nested PCR with universal phytoplasma 16S rRNA primers, and P1/P6 (Deng & Hiruki, 1991) and R16F2n/R16R2 primers (Gundersen & Lee, 1996), respectively. Amplicons of the expected size (1.5 Kb after direct PCR and 1.25 Kb after nested PCR) were obtained from diseased plants only. Nested PCR R16F2n/R16R2 products were Sanger sequenced in each direction, and the consensus sequence deposited in GenBank (Accession No. ON819760). BLASTn analysis revealed that the C. chinense phytoplasma 16Sr DNA sequence shared >99% sequence identity with those of the 16SrVI group including the isolate used to describe ‘Candidatus Phytoplasma trifolii’ (AY390261; Hiruki & Wang, 2004) and many strains found in India (e.g. MN861357 and MN861370). A phylogenetic tree (Figure 2) and an iPhyClassifier-based virtual RFLP analysis of the 16S rRNA gene sequence revealed that the phytoplasma-associated with little leaf of C. chinense belongs to the16SrVI-D subgroup with a similarity coefficient of 1.00. Previously, different phytoplasma subgroups have been associated with Capsicum annuum in India (‘Ca. P. asteris’, 16SrII-D; Sharma et al., 2015). In the present study, we confirmed the association of the ‘Ca. P. trifolii’ subgroup D of phytoplasmas with king chilli. The 16SrVI-D subgroup of phytoplasma has been associated with several other agricultural crops and weeds in northeast India (Dutta et al., 2022). To the best of our knowledge, this is the first report of the association of the 16SrVI-D phytoplasma subgroup with C. chinense and extends the known host range of this phytoplasma in India. In this study, affected crops had about 5–8% disease incidence based on visual observation and bearing in mind the symptoms observed, significant yield losses in commercially grown king chilli may occur. Further epidemiological and insect vector studies are needed to help formulate effective management strategies to prevent further spread.

Detection, characterization and management of brinjal little leaf disease in Assam

Aim: To record the prevalence, detection, molecular and biological characterization of phytoplasma associated with brinjal little leaf disease in Assam and its management. Methodology: Roving survey was conducted during the 2018-2020 brinjal growing seasons to record the disease incidence and symptoms associated with the disease. Insect and dodder transmission studies were carried out along with tetracycline tests. Further, molecular detection of all the samples collected during the survey and after dodder and insect transmission studies were done through PCR assays by using universal primers. Results: The disease incidence varied from 3.62 to 23.63 per cent in surveyed districts. Transmission studies revealed that Hishimonus phycitis (Dist.) is acting as a vector of brinjal little leaf phytoplasma and found to be transmitted by dodder (Cuscuta campestris). The phylogenetic analysis indicated that the Brinjal little leaf phytoplasma understudy was associated with ‘Candidatus phytoplasma trifolii’ and plants like Datura stramonium play a major role as potential alternate host of the phytoplasma. Further, studies on the effect of the antibiotic tetracycline hydrochloride in managing the disease elucidated that the antibiotic was effective only for temporary remission of symptoms. Interpretation: This study reveals a broad picture about the incidence, phytoplasma associated with the disease, putative leafhopper vector transmitting the phytoplasma in the field, alternate host harbouring the phytoplasma, and effect of antibiotic on the disease suppression, which will be useful to develop effective management strategies.

Hatay İli Bazı Kabak (Cucurbitaceae) ve Tarlasarmaşığı'nda (Convolvuluceae) Fitoplazmaların Belirlenmesi ve Karakterizasyonu

Aims: During a disease survey conducted in 2019, typical symptoms including yellowing of the leaves, stunting, shortening of internodes, proliferation of auxiliary shoots (witches'-broom), the bunchy appearance of growth at the ends of stems (rosetting), virescence of flowers or sterility, phyllody, small and deformed leaves and fasciation were observed on suspected cucurbit plants growing in Hatay province. Therefore, this study was carried out to determine the phytoplasma infection(s) on cucurbit crops and bindweed in Hatay province of Turkey.Methods and Results: In total; 4 pumpkins (winter squash: Cucurbita moschata Duchesne), 12 summer squash (Cucurbita pepo L.) and 2 bindweed (Convolvulus arvensis L.) samples were collected from suspected plants in cucurbit fields in Hatay province. Some of the plants (2 pumpkins, 10 summer squash and 2 bindweeds) exhibited symptoms related to phytoplasmas. Total nucleic acid was extracted by CTAB method. PCR amplification of 16S rDNA with phytoplasma specific primer pairs; F1/R0 followed by R16F2n/R2 confirmed the phytoplasma presence. Expected size amplicons of ~1.2 kb were obtained from infected samples from symptomatic cucurbits (12/12) and bindweed (1/2), but not from asymptomatic cucurbit plants (0/4). The sequences of the 4 symptomatic samples were deposited in NCBI GenBank (MT163353, MT163393, MT163396, and MT163469).Conclusions: After sequencing and phylogenetic analyses it was revealed that the squash isolates had 99% sequence identity with “Candidatus phytoplasma trifolii” (16SrVI) and the bindweed isolate had 99% sequence identity with “Candidatus phytoplasma solani” (16SrXII).Significance and Impact of the Study: There is a lack of knowledge on the status of phytoplasma infections and natural hosts in cucurbits in Turkey. According to our knowledge, this is the first report of a member of 16SrVI group, Candidatus phytoplasma trifolii associated with phytoplasma infection in winter squash-pumpkin (C. moschata) and summer squash (C. pepo) in Turkey.

First Report of 'Candidatus Phytoplasma trifolii' Associated with Little Leaf and Floral Virescence Disease in Solanum violaceum from the North East Region of India.

Solanum violaceum Ortega, (Indian nightshade), is a medicinal plant belonging to the family Solanaceae. It grows naturally as a weed in agricultural fields, forest edges, road sides and fallow lands throughout the North East Region of India. It is mostly consumed as a vegetable by the native people of the region because of its promising therapeutic effects (Islam and Islam 2018). In November 2019, typical phytoplasma-suspected symptoms such as little leaf, yellowing and floral virescence were observed on S. violaceum plants (Figure 1) in Kaliabor, Nagaon district of Assam, India (26.3220'N, 92.5540'E), with about 8% incidence based on visual observations. To investigate the possibility of a phytoplasma association with the symptoms, total DNA was isolated from collected leaf samples (symptomatic-6 and asymptomatic-9) by following the CTAB protocol (Kollar et al. 1990). The DNAs isolated were assayed for the presence of phytoplasma using polymerase chain reaction (PCR) assays performed with P1/P6 primer pair for 16S rRNA gene (Deng and Hiruki 1991) and SecAfor1/SecArev3 for secA gene (Hodgetts et al. 2008). The direct PCR targeting the 16S rRNA gene and secA gene amplified a product of about 1.5 kb and 840 bp, respectively, from all the symptomatic plant samples but not from any of the asymptomatic plant samples. All amplicons were double strand sequenced and corresponding high quality sequences were deposited in the GenBank with accession numbers MW261863 for 16S rRNA gene and MW885174 for secA gene with a sequence length of 1406 bp and 532 bp, respectively. Pairwise sequence comparison of 16S rRNA gene sequence of S. violaceum phytoplasma isolate revealed 100% sequence identity with strain of 'Candidatus Phytoplasma trifolii' (Accession number, EF186820) and secA gene showed up to 95% sequence identity with the same organism (Accession number, KX784498). Further analyses of the 16S rRNA and secA genes based phylogenetic tree (Figure 2A and B) and the iPhyClassifier-based virtual RFLP analysis of 16S rRNA gene revealed that the phytoplasma-associated with little leaf and floral virescence of S. violaceum belongs to 16SrIV-D subgroup with a similarity coefficient of 1.0. The 16S rRNA and secA gene sequences comparison confirmed the close association of phytoplasma strain associated with S. violaceum with 16SrVI-D subgroup phytoplasma. Earlier, the 16SrVI-D subgroup of phytoplasma has been reported to be associated with many horticultural and other agricultural crops in India (Rao 2021). Recently, the 16SrVI-D strain was reported in eggplant from Assam, India (Accession number, MW261866), showing up to 100% sequence identity with S. violaceum strain (Accession number, MW261863) (Dutta et al. 2020). To the best of our knowledge, this is the first report of the association of the 16SrVI-D subgroup of phytoplasma strain with S. violaceum plant, which is an important medicinal plant widely used in folk and traditional Indian systems of medicine. Since this study has confirmed S. violaceum as a new host of 16SrVI-D group of phytoplasma, further studies on the disease epidemiology and insects vectoring the phytoplasma would help formulate effective management strategies in preventing further spread to other hosts.

Identification of Melia azedarach and Eucalyptus camaldulensis as natural hosts and reservoirs of phytoplasmas in India

AbstractWitches' broom, leaf yellowing and little leaf symptoms were recorded on Melia azedarach in Pune district of Maharashtra and Dhanha, West Champaran district of Bihar. Little leaf and leaf yellowing symptoms also were recorded on Eucalyptus camaldulensis in Bhiwani district of Haryana. Symptomatic and asymptomatic leaf samples were collected from roadside trees for DNA extraction and PCR assay with phytoplasma‐specific primer pairs of 16S rRNA and secA genes. Amplicons of ~1.25 kb and ~840 bp were consistently obtained from all symptomatic samples of both the tree species using the phytoplasma‐specific primer pairs. Pairwise sequence analysis of 16S rRNA and secA gene sequences and phylogenetic analysis revealed that the phytoplasma strains associated with M. azedarach belonged to 16SrI‐B and 16SrVI‐D subgroups and those associated with E. camaldulensis were from the 16SrII‐D subgroup. This is the first report of an association of a ‘Candidatus Phytoplasma trifolii’‐related strain (16SrVI‐D subgroup) with M. azedarach globally and a ‘Ca. P. australasia’‐related strain with E. camaldulensis in India.

Lantana camara showing little leaf symptoms: A new weed host of ‘Candidatus Phytoplasma trifolii’

Lantana camara showing little leaf symptoms: A new weed host of ‘Candidatus Phytoplasma trifolii’

Detection, in silico analysis and molecular diversity of phytoplasmas from solanaceous crops in Turkey

Phytoplasma-like symptoms of leaf yellowing and calyx malformation were observed in eggplant (Solanum melongena L.), upward leaves and fruit malformation in pepper (Capsicum annuum L.), and aerial tuber formation in potato (S. tuberosum L.) during the survey performed in the late season (August to September) of 2015 and 2016 in Van province (Turkey). A total of 100 samples were tested by nested-PCR using universal primer pairs to assess the sanitary status of the solanaceous crops and to characterise the phytoplasma isolates. Among them, seven samples resulted in a 1.25 kb DNA fragment, and five (two eggplants, two peppers, and one potato) were molecularly characterised (Accession No.: KY579357, KT595210, MF564267, MF564266, and MH683601). BLAST and the virtual restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes revealed the presence of two distinct phytoplasma infections in solanaceous crops: ‘Candidatus Phytoplasma trifolii’ a member of the clover proliferation group (16SrVI) and subgroup A and ‘Candidatus P. solani’ a member of the stolbur group (16SrXII) and subgroup A. The virtual RFLP analysis and calculated coefficients of RFLP pattern similarities further revealed a remarkable genetic diversity among the ‘Candidatus P. solani’ isolates infecting pepper (similarity coefficient of 0.90) and eggplant (similarity coefficients of 0.98 and 1.00) at the same geographical area. This is the first report of the natural occurrence of ‘Candidadtus P. trifolii’ in potato from the Eastern Anatolia region, Turkey.

First report of a 'Candidatus Phytoplasma trifolii'-related strain (16SrVI-A) associated with white leaf disease in Spirea japonica

In August of 2018 phytoplasmas naturally infecting Spirea japonica were found in Şanlıurfa province, Turkey and they were identified by nested polymerase chain reaction (PCR), restriction fragment length polymorphism analysis (RFLP) and phylogeny based on sequence analysis. Plants exhibited typical white leaf disease symptoms with reduced leaf size. The phytoplasmas were found to be closely related to ‘Candidatus Phytoplasma trifolii’ (16SrVI-A). This paper has reported that Spirea japonica is a new host for ‘Ca. Phytoplasma trifolii’ which is associated with the white leaf disease in S. japonica.

First report of Candidatus Phytoplasma cynodontis (16SrXIV-A subgroup) associated with cauliflower phyllody and flat stem in India.

Symptoms of suspected phytoplasma infection were observed in cauliflower (Brassica oleracea var. botrytis) (cultivar NS60N) at Integrated Farming System Research Station, Trivandrum, Kerala, India (08o28'28"N, 76o57'47"E) in April-2021. The disease incidence was recorded up to 10% in different fields. The disease manifested as stunting, phyllody, floral malformation and flattening of stem (Fig.1A,B). Ten symptomatic and five asymptomatic plants were assayed for the presence of phytoplasma using nested PCR assays performed with P1/P7 and R16F2n/R16R2 primer pairs for 16S rRNA gene and SecAfor1/ SecArev3 and SecAfor2/ SecArev3 for secA gene (Deng and Hiruki 1991; Gundersen and Lee 1996; Hodgetts et al. 2008). The expected amplicons of ~1.25 kb and ~480 bp were consistently amplified in all the symptomatic cauliflower samples with the phytoplasma specific universal 16S rRNA and secA gene specific primers. Nested PCR products (~1.2 kb and 480 bp) amplified from cauliflower was cloned in EcoRI restriction sites of pGEM-T Easy vector (Promega, USA). The cloned nested PCR products were directly sequenced (16S rRNA gene: Acc. Nos. MZ196223, MZ196224; secA gene: MZ215721, MZ215722) in both forward and reverse directions which showed 99.77% sequence identity with Candidatus Phytoplasma cynodontis reference strain (Acc. No. AJ550984). Further analyses of the 16S rRNA and secA genes based phylogenetic tree (Fig. 2A and B) and the iPhyClassifier-based virtual RFLP analysis of 16Sr RNA gene study demonstrated that the phytoplasma-associated with cauliflower phyllody & flat stem disease (CaPP) belonged to 16SrXIV-A subgroup with a similarity coefficient of 1.0. No amplicon was observed from any of the asymptomatic cauliflower plants with the specific tested primers of both the genes. Earlier association of 16SrXV-A subgroup (Candidatus Phytoplasma brasiliense) and 16Sr III-J subgroup in Brazil (Canale and Badendo, 2013; Rappussi et al. 2012), 16SrII-A (Candidatus Phytoplasma aurantifolia) subgroup in China (Cai et al. 2016) and 16SrVI-A (Candidatus Phytoplasma trifolii) subgroup in Iran (Salehi 2007) were reported in cauliflower. Another species of cabbage, Brassica oleracea var. capitata L. was reported as host of Ca. P. trifloii (16Sr VI-D subgroup) from north India (Gopala et al. 2018). To our knowledge, this is the first report of a 'Candidatus Phytoplasma cynodontis', 16SrXIV-A subgroup related phytoplasma strain associated with cauliflower phyllody and flat stem in the world. The results described in this report confirm that the 16SrXIV-A phytoplasma, a widely distributed strain associated with sugarcane, wheat, grasses, sapota and many ornamentals in India (Rao 2021), has also infected cauliflower. This is not only the first instance of cauliflower phyllody disease found in India, but also the first instance of CaPP disease caused by 16SrXIV-A subgroup phytoplasma worldwide. This report has epidemiological significance and needs immediate attention, as cauliflower is the one of the most common vegetable crop grown all over India.

First Report of 'Candidatus Phytoplasma trifolii' Related Strain Associated with Yellowing and Witches'-Broom of Industrial Hemp (Cannabis sativa) in Arizona.

In Arizona, industrial hemp (Cannabis sativa) is a newly cultivated crop for fiber, oil, cosmetic products, and health food. During July to September 2020, two fields of industrial hemp crops were identified in southern Arizona with 10 to 30% incidence of plants showing witches' broom. Disease incidence was assessed by counting symptomatic plants in 4 randomly selected rows of 25 plants in each field. Symptoms ranged from leaf mottling and yellowing on mildly affected plants to leaf curling and shortened internode length of stem on severely affected plants (Fig. 1). Shoots were randomly collected from eight symptomatic plants and three asymptomatic plants in the same area. Genomic DNA was extracted from 200 mg of each sample using DNeasy Plant Pro Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. Phytoplasma was tested by a real-time PCR assay and TaqMan probe targeting the 23S ribosomal RNA gene that detects a wide range of known Phytoplasmas (Hodgetts et al., 2009). Beet curly top virus (BCTV) was targeted using BCTV-specific primers BCTV1 and BCTV2 following a method by Rondon (Rondon et al., 2016). BCTV was not detected in the plants, but Phytoplasmas were detected in all eight symptomatic plants, but not in the three control plants. The positive DNA samples were used to identify the phytoplasma by nested PCR using universal phytoplasma-specific primer pairs P1/P6 (Deng, S. et al. 1991) and R16F2n/R16R2 (Gundersen et al., 1996) targeting the 16S rRNA gene and the resulting 1.25 kb fragment in 4 positive samples was subjected to Sanger sequencing (Eton Bioscience, San Diego). All 4 sequences were identical and deposited in GenBank under accession MW981356. BLASTn results indicated 100% identity with that of several 'Candidatus Phytoplasma trifolii' strains on potato (KR072666, KF178706) in Washington and chile peppers (HQ436488) in New Mexico. It also shared 99.84% identity with the sequence of the reference strain of Candidatus Phytoplasma trifolii' (AY390261) that caused clover proliferation. The phytoplasma AZH1 was classified as a member of subgroup A within group16SrVI using iPhyClassifier, an interactive online tool for phytoplasma classification and taxonomic assignment (Zhao et al., 2013). Phylogenetic analysis revealed that the phytoplasma AZH1 clustered with other isolates of 'Candidatus Phytoplasma trifolii' (Fig. 2), including the strain NV1 associated with witches' broom on C. sativa in Nevada (Feng et al. 2019). This is the first report of 'Candidatus Phytoplasma trifolii' related strain associated with yellowing and witches' broom on hemp in Arizona. This finding is significant as the observation of symptoms at 30% incidence in one field suggested that the identified pathogen may pose a significant threat to the production of industrial hemp production in Arizona.

First report of 'Candidatus Phytoplasma trifolii' associated with leaf reddening and upright growth in pears (Pyrus communis L.)

The natural occurrence of 'Candidatus Phytoplasma trifolii' in pear trees (Pyrus communis Linnaeus) is reported here for the first time. In 2017, a total of thirty-five pear trees, two of them exhibiting leaf rolling along the midvein, reddening, bushy appearance, and upright growth symptoms were sampled in different locations in Van province, Turkey. The total deoxyribonucleic acid was extracted from symptomatic and asymptomatic plants. The purified DNA served as a template in nested polymerase chain reaction (nested-PCR) assays, performed to amplify 16S rRNA sequences using universal primer pairs (R16mF2/R16mR1 and R16F2n/R16R2). The resulting PCR products were then cloned into a pGEM T-Easy vector and sequenced bidirectionally. The phytoplasma strain, group, and subgroup identity were determined using the in silico restriction fragment length polymorphism (RFLP) analysis of the 16S ribosomal RNA-encoding gene sequences profiling with seventeen distinct restriction enzymes. Of the thirty-five pear samples, only two yielded 1 256 bp and 1 258 bp DNA fragments and were designated as Van-Pr3 (Acc. No. MH709141) and Van-Pr4 (Acc. No. MH730561), respectively. Based on the in silico virtual RFLP pattern analysis of the 16S rRNA sequences, we confirmed the presence of 'Ca. P. trifolii' belonging to the clover proliferation group and both identified phytoplasmas were identical with the similarity coefficient of 1.00 to the reference pattern of 16Sr group VI, subgroup A (Acc. No. AY390261). Here we report that the pear tree is an alternate host of the 'Ca. P. trifolii'.